U.S. patent application number 11/852609 was filed with the patent office on 2009-10-29 for alpha 1,4-galactosyltransferase and dna encoding thereof.
This patent application is currently assigned to Seikagaku Corporation. Invention is credited to Satoshi Fukumoto, Keiko Furukawa, Koichi Furukawa, Yoshinao KOJIMA, Tetsuya Okajima.
Application Number | 20090269802 11/852609 |
Document ID | / |
Family ID | 18559652 |
Filed Date | 2009-10-29 |
United States Patent
Application |
20090269802 |
Kind Code |
A1 |
KOJIMA; Yoshinao ; et
al. |
October 29, 2009 |
ALPHA 1,4-GALACTOSYLTRANSFERASE AND DNA ENCODING THEREOF
Abstract
The object of the present invention is to provide
.alpha.1,4-galactosyltransferase to transfer a galactose residue to
C4 position of galactose residue of lactosylceramide or
galactosylceramide, and DNA coding for the enzyme. What is provided
includes the following polypeptides (a) and (b), and DNAs encoding
thereof: (a) a polypeptide consisting of an amino acid sequence
represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or (b) a
polypeptide which comprises an amino acid sequence including
substitution, deletion, insertion or transposition of one or few
amino acids in the amino acid sequence of (a) and which has an
enzymatic activity to transfer a galactose residue from a galactose
donor to C4 position of galactose residue of lactosylceramide or
galactosylceramide which serves as an acceptor.
Inventors: |
KOJIMA; Yoshinao;
(Kariya-shi, JP) ; Fukumoto; Satoshi;
(Nishisonogi-gun, JP) ; Furukawa; Keiko;
(Nagoya-shi, JP) ; Okajima; Tetsuya; (Nagoya-shi,
JP) ; Furukawa; Koichi; (Nagoya-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Seikagaku Corporation
Tokyo
JP
|
Family ID: |
18559652 |
Appl. No.: |
11/852609 |
Filed: |
September 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10895131 |
Jul 21, 2004 |
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11852609 |
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09641701 |
Aug 21, 2000 |
6783966 |
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10895131 |
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Current U.S.
Class: |
435/69.1 ;
435/193; 435/320.1; 435/325; 435/97; 536/23.2 |
Current CPC
Class: |
C12P 19/58 20130101;
C12N 9/1051 20130101 |
Class at
Publication: |
435/69.1 ;
435/193; 536/23.2; 435/320.1; 435/325; 435/97 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 9/10 20060101 C12N009/10; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 5/06 20060101
C12N005/06; C12P 19/18 20060101 C12P019/18 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 2000 |
JP |
2000-035454 |
Claims
1. A polypeptide of (a) or (b) below: (a) a polypeptide consisting
of an amino acid sequence represented by the amino acid Nos. 46-353
in SEQ ID NO: 2; or (b) a polypeptide which comprises an amino acid
sequence including substitution, deletion, insertion or
transposition of one or few amino acids in the amino acid sequence
of (a) and which has an enzymatic activity to transfer a galactose
residue from a galactose donor to C4 position of galactose residue
of lactosylceramide or galactosylceramide which serves as an
acceptor.
2. A polypeptide of (a') or (b') below: (a') a polypeptide
consisting of an amino acid sequence represented by the amino acid
Nos. 20-353 in SEQ ID NO:2; or (b') a polypeptide which comprises
an amino acid sequence including substitution, deletion, insertion
or transposition of one or few amino acids in the amino acid
sequence of (a') and which has an enzymatic activity to transfer a
galactose residue from a galactose donor to C4 position of
galactose residue of lactosylceramide or galactosylceramide which
serves as an acceptor.
3. A polypeptide of (a'') or (b'') below: (a'') a polypeptide
consisting of an amino acid sequence represented by SEQ ID NO:2; or
(b'') a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a'') and which
has an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
4. A DNA encoding the polypeptide according to any one of claims 1
to 3.
5. The DNA according to claim 4 represented by (a) or (b) below:
(a) a DNA comprising a nucleotide sequence represented by
nucleotide Nos. 269 to 1192 in SEQ ID NO:1; or (b) a DNA
hybridizable with a DNA comprising a nucleotide sequence
represented by SEQ ID NO:1, a nucleotide sequence complimentary to
SEQ ID NO:1, or a part of those sequences, under a stringent
condition.
6. The DNA according to claim 5 encoding a polypeptide having an
enzymatic activity to transfer a galactose residue from a galactose
donor to C4 position of galactose residue of lactosylceramide or
galactosylceramide which serves as an acceptor.
7. A recombination vector containing the DNA as described in any
one of claims 4 to 6.
8. A transformed cell obtained by transfecting a host cell with the
DNA according to any one of claims 4 to 6, or the recombination
vector according to claim 7.
9. A method for producing the polypeptide according to any one of
claims 1 to 3, comprising the steps of: expressing the polypeptide
according to claims 1 to 3 by culturing the transformed cell
according to claim 8 in a medium suitable for expressing the
polypeptide; and recovering said polypeptide from the medium and/or
a cell extract of the cultured transformed cell.
10. A method for producing Gb3/CD77, comprising the steps of:
exposing the polypeptide according to any one of claims 1 to 3, or
a cultured product of the transformed cell according to claim 8, to
lactosylceramide, to cause thereby enzymatic reaction; and
recovering Gb3/CD77.
11. A method for producing a glycolipid represented by the
following formula (I), comprising the steps of: exposing the
polypeptide according to any one of claims 1 to 3, or a cultured
product of the transformed cell according to claim 8, to
galactosylceramide, to cause thereby enzymatic reaction; and
recovering the glycolipid represented by the following formula (I):
Gal.alpha.1.fwdarw.4Gal-Cer (1) wherein Gal represents a galactose
residue, Cer represents a ceramide residue and .alpha.1.fwdarw.4
represents an .alpha.1-4 glycosidic linkage.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the production of a
polypeptide using a recombinant DNA, or to a tool useful for
diagnosis or treatment of diseases, or more specifically to
.alpha.1,4-galactosyltransferase and DNA encoding thereof, a
recombination vector containing the DNA, and a transformed cell
transfected with the DNA or by the recombination vector, and to a
method for producing Gb3/CD77 or globo-series glycolipids by using
the transformed cell.
[0003] 2. Description of the Related Art
[0004] Glycosphingolipids are amphipathic molecules(ref.1) that are
synthesized by sequential actions of glycosyltransferases(ref.2).
Addition of one of three different sugars onto lactosylceramide
(which may be termed "LacCer" hereinafter) results in the synthesis
of either one of three major glycolipid series, i.e.,
ganglioside-series (a 2,3-sialic acid), lacto/neolacto-series
(.beta.1,4-N-acetylglucosamine) and globo-series
(.alpha.1,4-galactose). Although a number of genes coding for
enzymes responsible for the synthesis of the carbohydrate moiety of
glycosphingolipids have been recently isolated(ref.3), no
glycosyltransferase genes specific for the synthesis of
globo-series glycolipids have been isolated to date.
[0005] Globotriaosylceramide (hereinafter sometimes referred to as
"Gb3") is synthesized by .alpha.1,4-galactosyltransferase
(.alpha.1,4 Gal-T) from LacCer(ref.4). This glycolipid has been
characterized on red blood cells as the P.sup.k antigen of the P
blood group system(ref.5). Since Wiels et al(ref.6) reported that
Gb3 was a Burkitt's lymphoma associated antigen, the expression and
biological significance of Gb3 have been vigorously studied(ref.7,
8 and 9). Since Gb3 was clustered as CD77, this antigen will be
referred to as Gb3/CD77.
[0006] Gb3/CD77 was reported to be expressed in high amounts on
Burkitt's lymphoma cells. However, it is now considered to be a
differentiation antigen expressed on B cells, and can also be found
in some malignant tumors of B cell lineage(ref.7). Among normal
leukocytes, it is only expressed on a subset of tonsillar B cells
in the germinal centers (GC)(ref.9). Interestingly, GC B
lymphocytes expressing Gb3/CD77 undergo rapid and spontaneous
apoptosis when isolated and cultured in vitro(ref.11). Furthermore,
Burkitt's lymphoma cells with Gb3/CD77 antigen were also easily
induced to enter apoptosis upon culture at low serum concentration
or cross-linking by anti-immunoglobulin M antibodies(ref.12).
[0007] Gb3/CD77 has been recognized as a receptor for verotoxins
(VTs), the Shiga-like toxin from E. coli 0157 strain that can
trigger serious cytotoxic effects(ref.13 and 14). VT B-subunit
specifically binds to Gb3/CD77, then A subunit is incorporated into
cells, resulting in the degradation of 28 S ribosomal RNA and cell
death(ref.15). However, only B-subunit is also able to induce
apoptosis when cross-linked(ref.16). These results indicate that
Gb3/CD77 is a critical molecule in mediating apoptosis signals,
although the precise mechanisms remain to be investigated.
[0008] As stated above, it was revealed that Gb3/CD77, a
globo-series glycolipid, is synthesized by addition of
.alpha.1,4-galactose to LacCer(ref.4), but the glycosyltransferase
specific for this synethetic reaction has not been isolated yet. An
object of the present invention is to isolate a Gb3/CD77 synthase,
that is, a 1,4-galactosyltransferase, and DNA encoding thereof, and
to provide the use thereof.
[0009] The present inventors have studied hard to achieve the above
objects, and succeeded in isolating DNA encoding
.alpha.1,4-galactosyltransferase, revealing its nucleotide
sequence, and confirming that the DNA is responsible for the
expression of active .alpha.1,4-galactosyltransferase. Thus, they
achieved the present invention.
SUMMARY OF THE INVENTION
[0010] The present invention provides a polypeptide of (a) or (b)
below (hereinafter sometimes referred to as "the polypeptide of the
present invention"):
[0011] (a) a polypeptide consisting of an amino acid sequence
represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or
[0012] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
[0013] The polypeptide of the present invention also includes a
polypeptide of (a') or (b') below:
[0014] (a') a polypeptide consisting of an amino acid sequence
represented by the amino acid Nos. 20-353 in SEQ ID NO:2; or
[0015] (b') a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a') and which has
an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
[0016] The polypeptide of the present invention also includes a
polypeptide of (a'') or (b'') below:
[0017] (a'') a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO:2; or
[0018] (b'') a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a'') and which
has an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
[0019] The present invention also provides a DNA encoding the
polypeptides according to any one of above polypeptides
(hereinafter sometimes referred to as "the DNA of the present
invention"). The DNA of the present invention includes a DNA
represented by (a) or (b) below: [0020] (a) a DNA comprising a
nucleotide sequence represented by nucleotide Nos. 269 to 1192 in
SEQ ID NO:1; or [0021] (b) a DNA hybridizable with a DNA comprising
a nucleotide sequence represented by SEQ ID NO:1, a nucleotide
sequence complimentary to SEQ ID NO:1, or a part of those
sequences, under a stringent condition.
[0022] The DNA of the present invention also includes a DNA
encoding a polypeptide having an enzymatic activity to transfer a
galactose residue from a galactose donor to C4 position of
galactose residue of lactosylceramide or galactosylceramide which
serves as an acceptor.
[0023] The present invention still further provides a recombination
vector containing the DNA of the present invention.
[0024] The present invention still further provides a transformed
cell obtained by transfecting a host cell with the DNA of the
present invention or the recombination vector.
[0025] The present invention still further provides a method for
producing the polypeptide of the present invention, comprising the
steps of:
[0026] producing the polypeptide of the present invention by
culturing the transformed cell above in a medium; and
[0027] recovering said polypeptide from the medium and/or a cell
extract of the cultured transformed cell.
[0028] 10. The present invention still further provides a
method
[0029] for producing Gb/CD77 comprising the steps of: exposing the
polypeptide according to any one of claims 1 to 3, or a cultured
product of the transformed cell according to claim 8, to
lactosylceramide, to cause thereby enzymatic reaction; and
recovering Gb3/CD77.
[0030] The present invention still further provides a method for
producing a glycolipid as represented by the following formula (I)
comprising the steps of:
[0031] exposing the polypeptide of the present invention, or a
cultured product of the transformed cell above, to
galactosylceramide, to cause thereby enzymatic reaction; and
[0032] recovering the glycolipid represented by the following
formula (I):
Gal.alpha.1.fwdarw.4Gal-Cer (1)
wherein Gal represents a galactose residue, Cer represents a
ceramide residue and .alpha.1.fwdarw.4 represents an .alpha.1-4
glycosidic linkage.
[0033] In the present invention, the enzyme having an activity to
transfer a galactose residue from a galactose donor to C4 position
of galactose residue of lactosylceramide or galactosylceramide
which serves as an acceptor, will be called
".alpha.1,4-galactosyltransferase." Further, the activity of the
enzyme to transfer a galactose residue from a galactose donor to C4
position of galactose residue of lactosylceramide or
galactosylceramide which serves as an acceptor, will be called
".alpha.1,4-galactosyltransferase activity."
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows flow cytometry indicating the expression of
Gb3/CD77 by L cells. The left diagram relates to L cells
transfected with pCDM8 while the right diagram to L cells
transfected with pVTR1/CDM8. The thick line indicates the result of
cells stained with mAb38.13 and FITC-conjugated rabbit anti-rat IgG
(secondary antibodies) while the thin line the result of cells
stained only with the secondary antibodies (control).
[0035] FIG. 2 shows TLC charts of glycolipids extracted from cells
transiently transfected with .alpha.1,4 Gal-T gene.
[0036] A: TLC of glycolipids extracted from L cells transfected
with pCDM8 (VC) or pVTR1/CDM8 (TF). RBC represents neutral
glycolipids extracted from human B red blood cells.
[0037] B: TLC immunostaining of Gb3/CD77 by mAb38.13.
[0038] FIG. 3 shows the hydropathy plot of a polypeptide of the
present invention.
[0039] FIG. 4 shows the .alpha.1,4 Gal-T activity in the extracts
of transient transfectants of pVTR1.
[0040] A: .alpha.1,4 Gal-T activity when LacCer was used as an
acceptor.
[0041] B: .alpha.1,4 Gal-T activity when various acceptors were
used. PG represents paragloboside.
[0042] FIG. 5 shows the result of northern blotting of .alpha.1,4
Gal-T gene.
[0043] A: the upper columns show the results of hybridization with
a .sup.32P-labeled probe derived from pVTR1, while the lower
columns show the results of hybridization of the same membranes as
in A with a .beta.-actin cDNA probe (control).
[0044] B: the expression levels of mRNA of .alpha.1,4 Gal-T gene
were compared among various human tissues. The ordinate represents
the percentage of the expression level of a given tissue with
respect to the level of heart after correction with the
control.
[0045] FIG. 6 shows flowcytometry of stable transfectant cells. The
left diagram relates to cells transfected with pSV2neo while the
right diagram to cells transfected with pVTR1 and pSV2neo. The thin
line indicates the number of cells stained with mAb38.13 and
FITC-conjugated rabbit anti-rat IgG (secondary antibodies) while
the thick line the number of cells stained only with the secondary
antibodies (control).
[0046] FIG. 7 shows the results of MTT assay of L-neo and L-VTR1.
The left graph shows the result of L-neo while the right one the
result of L-VTR1.
[0047] FIG. 8 shows the effect of vero toxins on the cell
growth.
[0048] FIG. 9 shows an electrophoresis indicating the result of DNA
fragmentation assay.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] The mode for carrying out the present invention is described
below.
<1> The Polypeptide of the Present Invention
[0050] The polypeptide of the present invention is a polypeptide of
(a) or (b) below:
[0051] (a) a polypeptide consisting of an amino acid sequence
represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or
[0052] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
[0053] The above polypeptides contain at least a catalytic domain
of .alpha.1,4-galactosyltransferase as will be described later.
.alpha.1,4-galactosyltransferase comprises, in order from its
N-terminal, a cytoplasmic domain, transmembrane domain, and
catalytic domain. The polypeptides described above in (a') and (b')
comprise at least the transmembrane and catalytic domains. The
polypeptides described above in (a'') and (b'') comprise the
cytoplasmic, transmembrane and catalytic domains. These peptides
are all included in the polypeptides of the present invention.
[0054] An example of the amino acid sequence of a polypeptide of
the present invention is represented in SEQ ID NO:2. In SEQ ID
NO:2, amino acid Nos. 1-19 represents the cytoplasmic domain, Nos.
20-45 the transmembrane domain, and Nos. 46-353 the catalytic
domain.
[0055] Among those polypeptides, the polypeptides (a), (a') and
(a'') are preferred; the polypeptides (a') and (a'') are more
preferred; and the polypeptide (a'') is most preferred. However,
any one of them may be used as long as it has an
.alpha.1,4-galactosyltransferase activity.
[0056] In this specification, the term "a polypeptide which
comprises an amino acid sequence including substitution, deletion,
insertion or transposition of one or few amino acids and which has
an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an acceptor"
means that, one or more amino acid residues of the polypeptide may
be substituted, deleted, inserted, or transferred as long as such
modification does not substantially affect the ability to the
enzymatic activity (.alpha.1,4 Galactosyltransferase activity) of
the polypeptide to transfer a galactose residue from a galactose
donor to C4 position of galactose residue of lactosylceramide or
galactosylceramide which serves as an acceptor.
[0057] Mutation such as substitution, deletion, insertion, or
transposition of amino acid residues may occur in the amino acid
sequence of the polypeptides existing in nature due to, for
example, the modifying reaction of the biosynthesized polypeptides
in the living organisms or during their purification as well as
polymorphism and mutation of the DNAs encoding the polypeptides,
nevertheless some of mutated polypeptides are known to have
substantially the same physiological and biological activities as
the intact polypeptides that have not been mutated. The polypeptide
of the present invention includes those having slightly different
structures but not having a significant difference in the
functions. The polypeptide of the present invention also includes
those which have been artificially treated to have mutation as
described above in the amino acid sequences. In this case, a
further variety of mutants can be produced. For example, a
polypeptide having a human interleukin 2 (IL-2) amino acid
sequence, in which a cysteine residue has been replaced with a
serine residue, is known to retain the interleukin 2 activities
(Science 224, 1431 (1984)). Furthermore, a polypeptide of certain
kind is known to have a peptide region that is not essential for
exhibiting its activities. Examples of such polypeptides include a
signal peptide contained in a polypeptide that is secreted
extracellularly and a pro-sequence found in a precursor of
protease, and the like. Most of these regions are removed after
translation or upon conversion into an active form of the
polypeptides. These polypeptides exist in different primary
structures but finally have equivalent functions. Such polypeptides
are also included in the polypeptide of the present invention.
[0058] The term "few amino acids" used herein means the number of
amino acid residues that may be mutated to the extent that the
enzymatic activities of the polypeptide of the present invention
are not lost. For example, in a polypeptide consisting of 400 amino
acid residues, about 2 to 20, preferably 2 to 10, more preferably 2
to 5 or less of amino acid residues may be mutated.
[0059] The .alpha.1,4-galactosyltransferase activity can be assayed
by a known method(ref.4). Specifically, the assay consists of using
UDP-galactose (UDP-Gal) as a donor, and depending on the reaction
where galactose is transferred by the enzyme to Laccer (acceptor).
From above, it is obvious that any one skilled in the art could
easily select substitution, deletion, insertion or transposition of
one or more amino acid residues which does not substantially affect
the enzymatic activity, using its .alpha.1,4-galactosyltransferase
activity as an index.
[0060] The polypeptide of the present invention was obtained as
follows:
[0061] cDNA of .alpha.1,4-galactosyltransferase was isolated from
human melanoma cell line; the cDNA was expressed in mouse
fibroblasts; and the peptide was identified and characterized, and
its structure was determined. The polypeptide of the present
invention may be obtained by expressing the DNA of the present
invention as described later, in appropriate cells. The same
polypeptides chemically synthesized are naturally included in the
present invention. The method for producing the polypeptide of the
present invention using the DNA of the present invention will be
described later.
[0062] The polypeptide of the present invention is not necessarily
a single polypeptide but may be a part of a fusion protein if
necessary. A fusion protein comprising the polypeptide of the
present invention and another polypeptide such as protein A may be
cited as such an example.
[0063] The polypeptide of the present invention may consist of a
polypeptide alone, or contain a sugar chain or the like, as long as
it has the enzymatic activity to transfer a galactose residue from
a galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
<2> DNA of the Present Invention
[0064] The DNA of the present invention is a DNA encoding the
polypeptide of the present invention as described above. The DNAs
encoding the polypeptides described below in (a) and (b) may be
cited as an example:
[0065] (a) a polypeptide consisting of an amino acid sequence
represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or
[0066] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a galactose residue from a
galactose donor to C4 position of galactose residue of
lactosylceramide or galactosylceramide which serves as an
acceptor.
[0067] Among the DNAs, the one coding for polypeptide (a) is more
preferred.
[0068] The DNA encodes at least the catalytic domain of
.alpha.1,4-galactosyltransferase, but the DNA of the present
invention also includes DNAs encoding, in addition to above, the
polypeptides including a transmembrane domain and/or cytoplasmic
domain.
[0069] The DNA encoding the polypeptide (a) includes, for example,
a DNA containing a nucleotide sequence represented by nucleotide
Nos. 269-1192 in SEQ ID NO:1. The DNA encoding the polypeptide
containing the transmembrane domain includes, for example, a DNA
containing a nucleotide sequence represented by nucleotide Nos.
191-1192 in SEQ ID NO:1. The DNA encoding the polypeptide
containing the cytoplasmic domain includes, for example, a DNA
containing a nucleotide sequence represented by nucleotide Nos.
134-1192 in SEQ ID NO:1.
[0070] Furthermore the DNA comprising a nucleotide sequence
represented by SEQ ID NO:1 has been derived from human originally.
As a matter of course, however, the DNA of the present invention is
not limited to any source, and includes those that are produced by
genetic engineering procedure or chemical synthesis.
[0071] Furthermore, any one ordinarily skilled in the art would
readily understand that the DNA of the present invention includes
DNAs having nucleotide sequences different from what is described
above due to degeneracy of the genetic codes.
[0072] The DNA of the present invention also includes DNA or RNA
complementary to the DNA of the present invention. Furthermore, the
DNA of the present invention may be either a single-stranded coding
chain encoding the polypeptide of the present invention or a
double-stranded chain consisting of the above single-stranded chain
and a DNA or an RNA having a complementary nucleotide sequence
thereto.
[0073] The DNA of the present invention was obtained by expression
cloning as will be described later. However, since the nucleotide
sequence of the DNA of the present invention was determined, it
will be possible to isolate the same DNA from human-derived mRNA or
cDNA, or a chromosomal DNA through PCR with an oligonucleotide
prepared from the nucleotide sequence thus determined to serve as a
primer, or from a cDNA library or chromosomal DNA library through
hybridization with an oligonucleotide prepared from the nucleotide
sequence thus determined to serve as a probe.
[0074] The gene encoding the polypeptide of the present invention
derived from a chromosome is expected to contain introns in the
coding region. DNA fragments separated by introns are also included
in the DNA of the present invention.
[0075] The DNA of the present invention may include DNAs, as long
as they code for the polypeptides having an enzymatic activity to
transfer a galactose residue from a galactose donor to C4 position
of galactose residue of lactosylceramide or galactosylceramide
which serves as an acceptor, hybridizable with a probe comprising a
nucleotide sequence complimentary to the nucleotide sequence of
SEQ. ID No:1, or to a nucleotide sequence represented by nucleotide
Nos. 269-1292, nucleotide sequence represented by nucleotide Nos.
191-1292, or nucleotide sequence represented by nucleotide Nos.
134-1292 of SEQ ID No:1, or with a probe comprising a part of those
nucleotide sequences, under a stringent condition. The "stringent
condition" here refers to a condition under which a so-called
specific hybrid is formed, but no non-specific hybrids are formed
(see Sambrook, J. et al., Molecular Cloning A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press (1989)). The
"stringent condition" may include for example subjecting a test DNA
to a solution containing 50% formamide, 4.times.SSC, 50 mM HEPES
(pH 7.0), 10.times.Denhardt's solution, and 100 .mu.g/ml of sermon
sperm DNA, allowing it to hybridize in the solution at 42.degree.
C., and washing the yield in 2.times.SSC and 0.1% SDS solution at
room temperature, then in 0.1.times.SSC and 0.1% SDS solution at a
temperature of 50.degree. C. or less.
[0076] Production of the polypeptide of the present invention may
be achieved by cultivating transformed cells transfected with the
DNA of the present invention on appropriate growth medium, thereby
allowing the polypeptide of the present invention encoded by the
DNA of the present invention to express itself, and by recovering
the polypeptide thus expressed. The thus expressed polypeptide of
the present invention can be extracted from a cultured product of
the transformed cells (comprising both transformed cells and
medium). However, if the polypeptide of the present invention is
accumulated in the cytoplasm of transformed cells, or their
membrane fraction, the polypeptide must be extracted from the
transformed cells. Or, if the polypeptide is accumulated in medium,
it must be extracted from medium. Or, if use of the transformed
cells in which the polypeptide is expressed is desired, the
transformed cells themselves, or their processed products may be
used intact, or after they have been bound to an appropriate solid
phase, or covered with gel for solidification. The "transformed
cell" includes not only transformed cell themselves but also
extracts from them.
[0077] For the transfection of the DNA of the present invention
into a host cell, it is only necessary to prepare a recombination
vector by inserting the DNA of the present invention into an
appropriate vector, and to introduce the DNA of the present
invention into a host cell through the recombination vector. The
vector is preferably an expression vector.
[0078] The host cell is not limited to any specific cells, as long
as they can fully play the function of the DNA of the present
invention, or of the recombination vector containing the DNA of the
present invention. Thus, it may include any animal cells, plant
cells, micro-organisms (bacteria), or the like. Procaryotic cells
such as E. coli, or eucaryotic cells such as mammalian cells may be
exemplified. When a procaryotic cell such as E. coli is used,
addition of sugar chain does not occur to the polypeptide produced
as by expression of the DNA of the present invention, then the
polypeptide of the present invention having no sugar chain can be
obtained. When eucaryotic cell such as a mammalian cell is used,
sugar chain may add to the polypeptide produced by expression of
the DNA of the present invention, then the form of the polypeptide
of the present invention comprising sugar chain can be
obtained.
[0079] Specifically, the host cell to transfect with the DNA of the
present invention may include for example L cells derived from
mouse fibroblasts. Specifically, the vector may include pCDM8 or
pcDNA3 expression vector (both available from Invitrogen). The
culture medium and condition may be chosen appropriately according
to a given host cell.
[0080] The DNA of the present invention may be expressed directly.
Alternatively, it may be expressed with another polypeptide as a
fusion polypeptide. The full-length DNA of the present invention
may be expressed. It may also be expressed in part as a partial
peptide.
[0081] The method for introducing the DNA of the present invention
may depend on transfection based for example on DEAE-dextran
method.
[0082] Recovering the polypeptide of the present invention from a
cultured product may be performed by known extraction and
purification methods for polypeptides. The cultured product used
herein includes the medium and the cells in the medium.
[0083] Extraction of the polypeptide of the present invention may
be performed, for example, by a method using a nitrogen cavitation
apparatus, extraction from the cells disrupted by homogenization,
glass bead milling, sonic wave treatment, osmotic shock,
freeze-thawing procedure or the like, extraction by using
detergent, or combination of those methods.
[0084] When the DNA of the present invention encoding the
polypeptide (a'') is expressed in L cells, the polypeptide of the
present invention is localized at the membrane fraction of the
cell. When the DNA of the present invention is expressed as a
fusion protein comprising a polypeptide of the present invention
(or a part thereof), and another peptide, so as to be a soluble
protein, that fusion protein may be present in the cytoplasm. When
the DNA of the present invention is expressed as a fusion protein
comprising the polypeptide of the present invention or a part
thereof and a secretion signal, the resulting protein may be
secreted into medium. Isolation of DNA encoding a part of the
polypeptide of the present invention may be achieved by preparing a
primer previously designed to produce such a DNA, and applying the
primer in PCR to human derived mRNA, cDNA library, or chromosomal
DNA.
[0085] Specific examples of the method of purifying the polypeptide
of the present invention extracted from the cells or medium include
salting out with salt such as ammonium sulfate or sodium sulfate,
centrifugation, dialysis, ultrafiltration, absorption
chromatography, ion exchange chromatography, hydrophobic
chromatography, reverse phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, and any combination thereof.
[0086] It can be confirmed whether the polypeptide of the present
invention has been produced or not by analyzing amino acid
sequence, action, and substrate specificity of the purified
polypeptide.
<3> Utilization of the Polypeptide and DNA of the Present
Invention
[0087] The polypeptide of the present invention can be utilized for
the synthesis of globo-series glycolipids, for example, for the
synthesis of Gb3/CD77 by exposing the polypeptide of the present
invention or a cultured product of the transformed cells
transfected with the DNA of the present invention to
lactosylceramide, thereby evoking enzymatic reaction. Also, it is
possible to obtain a glycolipid as represented by the following
formula (I) by exposing the polypeptide of the present invention or
a cultured product of the transformed cells transfected with the
DNA of the present invention to galactosylceramide.
Gal .alpha.1.fwdarw.4Gal-Cer (1)
[0088] In this procedure, exposure to substrate may occur through
contact with the transformed cells, if the polypeptide of the
present invention is produced and accumulated in the cytoplasm or
in the membrane fraction, or through contact with medium if the
polypeptide is accumulated in medium. When the cells in which the
polypeptide of the present invention has been expressed are
utilized, exposure to substrate may occur through direct contact
with the cells themselves, or extracts therefrom, or immobilized
extracts. The term "transformed cell" here includes not only
transformed cells themselves but also extracts from them.
[0089] The polypeptide of the present invention is capable of
specifically attaching a galactose residue to C4 position of
galactose residue of lactosylceramide or galactosylceramide
contained in a sugar chain of a glycoprotein. Further, the
polypeptide of the present invention is utilized for selective
synthesis of a sugar chain.
[0090] Although a number of members of
.beta.1,3-galactosyltransferases (.beta.1,3Gal-Ts) or
.beta.1,4Gal-Ts have been identified(ref.27-30), this gene is the
first and only .alpha.1,4Gal-T gene isolated so far. Moreover, no
homologous genes to this gene were detected in the data base of C.
elegans or Drosophila melanogaster genes, even though many
.beta.1,4 and .beta.1,3Gal-T-related genes have been identified.
These facts may indicate that .alpha.1,4Gal-T gene evolved
relatively later than other galactosyltransferase genes, and
globo-series glycolipids synthesized through Gb3 are playing more
precise roles compared to glycolipids of the other series.
[0091] Gb3/CD77 seems to be unusual because it can mediate various
apoptotic signals in both normal cells and malignant tumor cells,
even though it does not contain any cytoplasmic domain(ref.16 and
31). The observed rapid death of CD77.sup.+ BC B cells in vitro
suggests that endogenous ligand molecules interact with Gb3/CD77 to
bring about the physiologic selection of immature B cells(ref.11
and 32). Furthermore, the capability of B subunit of VT to induce
apoptosis of Gb3/CD77.sup.+ cells(ref.16) strongly encourages the
investigation of Gb3/CD77-associating cytoplasmic
molecules(ref.31). Investigations of these ligands and signal
transducers relevant to Gb3/CD77 might contribute to further
understanding of the B cell selection and of the pathogenesis of
hemolytic uremic syndrome caused by E. coli 0157 infection. In
particular, the tissue specificity of the syndrome such as renal
failure, hemolysis and neurological disorders, might be clarified
by gene manipulation of the cloned Gb3/CD77 synthase.
[0092] Furthermore, it has recently been reported that Gb3/CD77 and
ganglioside GM3 may function as alternative cofactors for the entry
of human immunodeficiency virus type 1 (HIV-1) in CD4-induced
interactions between gp120 and glycosphingolipid
microdomains(ref.33 and 34). If this is the case, Gb3/CD77 may be a
receptor not only for bacterial toxins but for viruses, and the
regulation of Gb3/CD77 expression could be a key target for the
therapeutic approaches of viral infections such as HIV-1.
[0093] Further, the expression of Gb3/CD77 in the kidney has been
thought to be related with the development of hemolytic uremic
syndrome (HUS). Furthermore, Fabry's disease is known as a disease
in which Gb3/CD77 accumulates in the kidney, heart, brain and
vasculature. From above, the polypeptide and DNA of the present
invention, and the method of the present invention for producing
Gb3/CD77 or a glycolipid may serve as a therapeutic tool,
diagnostic tool or research tool for the treatment of diseases
caused by the abnormal expression of Gb3/CD77 as described
above.
EXAMPLE
[0094] The present invention will be described more in detail below
by means of examples.
Example 1
Isolation of cDNA for .alpha.1,4-Galactosyltransferase (.alpha.1,4
Gal-T)
[0095] <1> Preparation of a cDNA Library from a Human
Melanoma Cell Strain, and Cloning of .alpha.1,4 Gal-T cDNA.
[0096] A cDNA was prepared from poly(A.sup.+) RNA of a human
melanoma cell line SK-MEL-37 as described(ref.17). The cDNA library
was constructed by inserting the cDNA into a vector plasmid pCDM8
(Invitrogen). The library contained 5.times.10.sup.6 independent
colonies. The strain of bacteria was E. coli MC1061/P3
(ref.18).
[0097] Since the SK-MEL-37 cell line does not express Gb3/CD77 on
its surface, it highly efficiently expresses .alpha.1,4 Gal-T, and
thus the cDNA library prepared from this cell line is excellent for
the present purpose.
[0098] Plasmids of the cDNA library were transfected into a mouse
fibroblast L cells together with pd13027 (polyoma T gene, provided
by Dr. C. Basilico at New York University, New York) using
DEAE-dextran as described (ref.18). L cells express a large amount
of LacCer although they have no .alpha.1,4 Gal-T activity nor
Gb3/CD77 expression(ref.19). The L cells, because of these
characteristics, served as an excellent host in the cloning of cDNA
for .alpha.1,4 Gal-T. The L cell was kindly provided by Dr. A. P.
Albino at Sloan-Kettering Cancer Center, New York, and was
maintained in Dulbecco's modified Eagle's minimal essential medium
(DMEM) containing 7.5% of fetal bovine serum (FCS).
[0099] After 48 h, the transfected cells were detached and
incubated with a rat monoclonal antibody (mAb) 38.13 (ref.6) on ice
for 45 min. After washing, cells were plated on dishes coated with
rabbit anti-rat IgM (ZYMED) as described (ref.17). Plasmid DNA was
rescued from the panned cells by preparing Hirt extracts, and
transformed into MC1061/P3. The same procedure was repeated 5
times. The plasmid DNA was collected from the transformed
cells.
[0100] Using microscale transfection of L cell and
immunofluorescence assay, cDNA clones that determined the Gb3/CD77
expression were isolated. Cell surface expression of Gb3/CD77 was
analyzed by flow cytometry (Becton Dickinson) as described
(ref.19). MAbs 38.13 or TU-1 (23) were used with FITC-conjugated
rabbit anti-rat IgG or anti-mouse IgM (ZYMED), respectively. As a
result, two clones showing positive reactions were successfully
isolated. As described later, the two clones are essentially
similar in nature, and thus one of them is called pVTR1, and
further analysis was performed on that one.
[0101] FIG. 1 shows the results of flow cytometry of L cells
transfected with the plasmid pVTR1 or the plasmid pCDM8 (containing
no target sequence to serve as control). It is obvious from this
that the cells transfected with pVTR1 express Gb3/CD77 while those
transfected with pCDM8 alone do not express Gb3/CD77. It was thus
demonstrated that .alpha.1,4 Gal-T cDNA inserted to pVTR1 is
involved in the synthesis of Gb3/CD77.
<2> Extraction of Glycolipids from the Transformed Cells, and
Identification of Gb3/CD77
[0102] Glycolipids were extracted as described(ref.21). Briefly,
glycolipids were extracted from about 400 .mu.l of packed cells
using chloroform/methanol (2:1, 1:1, 1:2) sequentially. TLC was
performed on a high performance TLC plates (MERCK, Darmstadt) using
the solvent system chloroform:methanol:0.22% CaCl.sub.2 (60:35:8)
and sprayed by orcinol. For standards, bovine brain ganglioside
mixture (Wako, Tokyo), neutral glycolipids from human erythrocytes,
and Gb3 (Sigma) were used.
[0103] Glycosphingolipids extracted from the transformed cells
showed definite Gb3 bands in TLC, although the transformed cells
with pCDM8 alone showed no Gb3 band (FIG. 2A), suggesting that the
cloned pVTR1 derived from .alpha.1,4Gal-T gene.
[0104] The identity of Gb3/CD77 was confirmed by TLC-immunostaining
using an aluminum-backed silica plate (MERCK) as described(ref.21).
After TLC, the plate was blotted onto PVDF membrane as
described(ref.22). After blocking, the plate was incubated with
mAb, then antibody binding was detected with ABC kit (Vector
Laboratories, Burlingame, Calif.) and Konica Immunostaining
HRP-1000 (Konica, Tokyo). This TLC-immunostaining revealed strong
bands of Gb3 only in the extracts from the cDNA transfected cells
(FIG. 2B).
<3> Nucleotide Sequencing of Gene for .alpha.1,4 Gal-T
[0105] The nucleotide sequence of the cDNA clone which was
confirmed to express Gb3/CD77 as described above was determined by
dideoxynucleotide termination sequencing using the PRISM dye
terminator cycle sequencing kit and model 310 DNA sequencer
(Applied Biosystems). The sequencing showed that the two clones are
essentially the same in sequence. Accordingly, one of them was
selected for subsequent analysis, and named pVTR1. The nucleotide
sequence of cDNA in pVTR1 is shown by SEQ ID NO:1. The amino acid
sequence encoded by this nucleotide sequence is shown by SEQ ID
NOs:1 and 2.
[0106] The initiation codon is embedded within a sequence similar
to the Kozak consensus initiation sequence(ref. 24 and 25). This
open reading frame predicts a 353-amino acid protein with a
molecular mass of 40,498 daltons.
[0107] Nucleotide and amino acid sequence homology search was
carried out using the internet program BLAST (National Center for
Biotechnology Information). However, no cDNA or protein having a
high homology with these sequences was found in the database.
[0108] Amino acid sequence and hydropathy analyses (35) were
performed with a software GENETYX-MAC version 8.0 (Software
Development, Tokyo)(FIG. 3). A single hydrophobic segment with 26
amino acids was present near the amino terminus (amino acid Nos.
20-45 in SEQ ID NO: 2). This putative signal anchor sequence would
place 19 residues within the cytoplasm and 308 amino acids within
the Golgi lumen.
[0109] The presence of two potential N-glycosylation sites are
indicated (amino acid sequence Nos. 121-123 and 203-205 in SEQ ID
NO: 2). Relatively high frequency of proline (10/31) was detected
at the C'-side of the transmembrane domain.
Example 2
Characterization and Production of .alpha.1,4 Gal-T
<1> Enzyme Assay of .alpha.1,4 Gal-T
[0110] Membrane fractions were prepared as described(ref.19) from L
cells transfected with the gene for .alpha.1,4 Gal-T as obtained in
Example 1. The enzyme activity of .alpha.1,4 Gal-T in the membrane
fraction was measured as described previously(ref.4). The reaction
mixture for the assay contained the following in a volume of 50
.mu.l: 50 mM sodium cacodylate-HCl (pH 6.0), 10 mM MgCl.sub.2, 5 mM
galactonolactone (Sigma), 0.3% Triton X-100 (Sigma), 0.4 mM
(LacCer), 2.9 mM phosphatidylglycerol (Sigma), 0.2 mM UDP-Gal
(Sigma), UDP-[.sup.14C] Gal (2.5.times.10.sup.5 dpm) (NEN), and
membrane fraction containing 50 .mu.g protein. The protein
concentration was determined by Lowry's methods(ref.20). The
products was isolated by a C.sub.18 Sep-Pak cartridge (Waters,
Milford, Mass.) and analyzed by thin layer chromatography (TLC) and
autoradiography using a Bio-Imaging Analyzer BAS2000 (Fuji Film,
Tokyo). The results are shown in FIG. 4A.
[0111] L cells transfected with pVTR1/CDM8 showed high Gb3 synthase
activity (7,012 units, pmol/h/mg of protein) when LacCer was used
as an acceptor. On the other hand, L cells transfected with pCDM8
alone were completely negative. Thus, this cDNA determined
.alpha.1,4Gal-T activity and the surface expression of Gb3/CD77,
indicating that this cDNA encodes the Gb3/CD77 synthase.
[0112] Enzyme activity toward other potential acceptors was also
examined (FIG. 4B). None of the acceptors examined except LacCer
and galactosylceramide showed significant levels of
[.sup.14C]galactose incorporation (FIG. 4B). Km values for these
two substrates were 54.5 .mu.M (LacCer) and 132 .mu.M
(galactosylceramide). The P1 antigen in the P blood group system is
also formed by .alpha.1,4 galactose transfer (acting on
paragloboside(PG)), but it was confirmed that this enzyme is not
responsible for the synthesis of P1 antigen (FIG. 4B).
Example 3
Expression Analysis of .alpha.1,4 Gal-T Gene in Various Tissues
(Northern Blotting)
[0113] Multiple Choice Northern Blots membranes (OriGene
Technologies, Rockville, Mass.) were used. They were hybridized
with [.sup.32P]dCTP-labeled cDNA probe of pVTR1 or control
.beta.-actin as described (ref.18 and 19). The relative expression
levels of mRNA of .alpha.1,4Gal-T gene among human tissues measured
by Bio-Imaging Analyzer BAS2000 (Fuji Film) are presented as a
percentage of the value of heart after correction with the control.
Expression levels of the al, 4Gal-T gene in various human tissues
were examined by Northern blotting. Among tissues examined, heart,
kidney, spleen, liver, testis and placenta strongly expressed the
gene (FIG. 5).
Example 4
Stable Transfection of Cells with pVTR1 Plasmid
[0114] To prepare stable transformants, pVTR1 and pSv2neo were
co-transfected into L cells using Lipofection kit (TOYOBO, Tokyo,
Japan). To select transformants, the cells were cultured in DMEM
containing FCS (7.5%) and G418 (300 .mu.g/ml). G481 is inactivated
by 3'-O-aminoglycoside phosphotransferase encoded by the neo
gene.
[0115] G418-resistant cells were cloned by limiting dilution.
Clones transfected with pSv2neo alone were prepared for control.
These cells were incubated together with mAb38.13, followed by
addition of FITC-conjugated rabbit anti-rat IgG for reaction, and
the resulting cells were subjected to flow cytometry in the same
manner as described above. The results showed that the cells
transfected with pVTR1 and pSV2neo(L-VTR1) strongly expressed
Gb3/CD77 while those transfected with pSV2neo alone(L-neo) did not
express Gb3/CD77 (FIG. 6).
Example 5
Reaction of Transformed Cells to Verotoxins
<1> MTT Assay
[0116] To compare the reactions of L-VTR1 and L-neo to VTs, MTT
assay was performed using cells prepared in 48 well plates
(1.times.10.sup.4 cells/well) and cultured in the presence of VT1
or VT2. The assay was performed by triplicated samples. To quantify
the cell proliferation, 50 .mu.l of 5 mg/ml of MTT (Sigma) in PBS
was added to each well.
[0117] After incubation for 5 h at 37.degree. C., the supernatants
were aspirated and 100 .mu.l of n-propylalchohol containing 0.1%
NP40 and 4 mM HCl was added. The color reaction was quantitated
using automatic plate reader IMMUNO-MINI NJ-2300 (Nihon InterMed,
Tokyo, Japan) at 590 nm with a reference filter of 620 nm.
[0118] L-VTR1 in VT (+) medium showed marked growth suppression
compared to that cultured in the absence of VT, while L-neo showed
no effects of VT (FIG. 7). MTT assay of L-VTR1 and L-neo after the
exposure to various concentrations of VTs revealed marked growth
suppression of L-VTR1 even at 0.01 ng/ml, but not of L-neo (FIG.
8).
<2> DNA Fragmentation Assay
[0119] DNA fragmentation assay was performed to determine the
mechanism responsible for the death of L-VTR1 treated with VTs.
Cells were cultured in the presence of VT2 (200 ng/ml). After 24 h,
cells were collected and the pellets were lysed in 100 .mu.l of
lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM EDTA and 0.5% Triton
X-100) for 10 min at 4.degree. C. After centrifugation, the
supernatants were collected, and 2 .mu.l of RNAse (10 mg/ml) and 2
.mu.l of Proteinase K (10 mg/ml) were added. After incubation for 1
h at 37.degree. C., the fragmented DNA was 2-propanol precipitated.
Electrophoresis was conducted using DNA derived from
1.5.times.10.sup.6 cells in 2% agarose gel containing 0.2 .mu.g/ml
ethidium bromide in TEA buffer.
[0120] Agarose gel electrophoresis of cytoplasmic DNA extracted
from L-VTR1 revealed a clear pattern of DNA fragmentation
characteristic of apoptosis (FIG. 9). In contrast, the L-neo sample
did not show any ladder formation. Thus, it was confirmed that
Gb3/CD77 generated by the cDNA serves as a functional receptor for
VTs.
[0121] The present invention provides
.alpha.1,4-galactosyltransferase, and DNA encoding thereof. That
enzyme can be utilized for the production of globo-series
glycolipids such as Gb3/CD77.
[0122] Further, the DNA is useful for production of the above
described enzyme, or serves as a therapeutic tool, diagnostic tool
or research tool for the treatment of diseases caused by the
abnormal expression of Gb3/CD77, or it may be useful for the
treatment or diagnosis of diseases involved in the action of
verotoxins.
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Sequence CWU 1
1
211975DNAHomo sapiensCDS(134)..(1192) 1aaggtcggct gctgagccag
ggcgtgtctc ccggaggcct gtgggctgcc aggatcccca 60cctctctgca atgggctgcc
caggctgacc agccggttcc tgctggaagc tcctggtctg 120atctggggat acc atg
tcc aag ccc ccc gac ctc ctg ctg cgg ctg ctc 169 Met Ser Lys Pro Pro
Asp Leu Leu Leu Arg Leu Leu 1 5 10cgg ggc gcc cca agg cag cgg gtc
tgc acc ctg ttc atc atc ggc ttc 217Arg Gly Ala Pro Arg Gln Arg Val
Cys Thr Leu Phe Ile Ile Gly Phe 15 20 25aag ttc acg ttt ttc gtc tcc
atc atg atc tac tgg cac gtt gtg gga 265Lys Phe Thr Phe Phe Val Ser
Ile Met Ile Tyr Trp His Val Val Gly 30 35 40gag ccc aag gag aaa ggg
cag ctc tat aac ctg cca gca gag atc ccc 313Glu Pro Lys Glu Lys Gly
Gln Leu Tyr Asn Leu Pro Ala Glu Ile Pro45 50 55 60tgc ccc acc ttg
aca ccc ccc acc cca ccc tcc cac ggc ccc act cca 361Cys Pro Thr Leu
Thr Pro Pro Thr Pro Pro Ser His Gly Pro Thr Pro 65 70 75ggc aac atc
ttc ttc ctg gag act tca gac cgg acc aac ccc aac ttc 409Gly Asn Ile
Phe Phe Leu Glu Thr Ser Asp Arg Thr Asn Pro Asn Phe 80 85 90ctg ttc
atg tgc tcg gtg gag tcg gcc gcc aga act cac ccc gaa tcc 457Leu Phe
Met Cys Ser Val Glu Ser Ala Ala Arg Thr His Pro Glu Ser 95 100
105cac gtg ctg gtc ctg atg aaa ggg ctt ccg ggt ggc aac gcc tct ctg
505His Val Leu Val Leu Met Lys Gly Leu Pro Gly Gly Asn Ala Ser Leu
110 115 120ccc cgg cac ctg ggc atc tca ctt ctg agc tgc ttc ccg aat
gtc cag 553Pro Arg His Leu Gly Ile Ser Leu Leu Ser Cys Phe Pro Asn
Val Gln125 130 135 140atg ctc ccg ctg gac ctg cgg gag ctg ttc cgg
gac aca ccc ctg gcc 601Met Leu Pro Leu Asp Leu Arg Glu Leu Phe Arg
Asp Thr Pro Leu Ala 145 150 155gac tgg tac gcg gcc gtg cag ggg cgc
tgg gag ccc tac ctg ctg ccc 649Asp Trp Tyr Ala Ala Val Gln Gly Arg
Trp Glu Pro Tyr Leu Leu Pro 160 165 170gtg ctc tcc gac gcc tcc agg
atc gca ctc atg tgg aag ttc ggc ggc 697Val Leu Ser Asp Ala Ser Arg
Ile Ala Leu Met Trp Lys Phe Gly Gly 175 180 185atc tac ctg gac acg
gac ttc att gtt ctc aag aac ctg cgg aac ctg 745Ile Tyr Leu Asp Thr
Asp Phe Ile Val Leu Lys Asn Leu Arg Asn Leu 190 195 200acc aac gtg
ctg ggc acc cag tcc cgc tac gtc ctc aac ggc gcg ttc 793Thr Asn Val
Leu Gly Thr Gln Ser Arg Tyr Val Leu Asn Gly Ala Phe205 210 215
220ctg gcc ttc gag cgc cgg cac gag ttc atg gcg ctg tgc atg cgg gac
841Leu Ala Phe Glu Arg Arg His Glu Phe Met Ala Leu Cys Met Arg Asp
225 230 235ttc gtg gac cac tac aac ggc tgg atc tgg ggt cac cag ggc
ccg cag 889Phe Val Asp His Tyr Asn Gly Trp Ile Trp Gly His Gln Gly
Pro Gln 240 245 250ctg ctc acg cgg gtc ttc aag aag tgg tgt tcc atc
cgc agc ctg gcc 937Leu Leu Thr Arg Val Phe Lys Lys Trp Cys Ser Ile
Arg Ser Leu Ala 255 260 265gag agc cgc gcc tgc cgc ggc gtc acc acc
ctg ccc cct gag gcc ttc 985Glu Ser Arg Ala Cys Arg Gly Val Thr Thr
Leu Pro Pro Glu Ala Phe 270 275 280tac ccc atc ccc tgg cag gac tgg
aag aag tac ttt gag gac atc aac 1033Tyr Pro Ile Pro Trp Gln Asp Trp
Lys Lys Tyr Phe Glu Asp Ile Asn285 290 295 300ccg gag gag ctg ccg
cgg ctg ctc agt gcc acc tat gct gtc cac gtg 1081Pro Glu Glu Leu Pro
Arg Leu Leu Ser Ala Thr Tyr Ala Val His Val 305 310 315tgg aac aag
aag agc cag ggc acg cgg ttc gag gcc acg tcc agg gca 1129Trp Asn Lys
Lys Ser Gln Gly Thr Arg Phe Glu Ala Thr Ser Arg Ala 320 325 330ctg
ctg gcc cag ctg cat gcc cgc tac tgc ccc acg acg cac gag gcc 1177Leu
Leu Ala Gln Leu His Ala Arg Tyr Cys Pro Thr Thr His Glu Ala 335 340
345atg aaa atg tac ttg tgaggggccc gccaggtcac ctccccaacc tgctcctgat
1232Met Lys Met Tyr Leu 350ggggcactgg gccgcccttc ccggggaggc
aagattgagg gcccgggaga gggaggcccg 1292agctgccacc gggcttaggc
aggctgttga ggagctgtgg gagcaggccc agtgggaggc 1352tgtggacacc
ccgaggacag tgtcctgtct cgaggcaggg ctgacacatg gtgccatagc
1412cagcggaggg cgctcagtga gtgccccggg ccttctagac aacaggcagg
aaggatgaac 1472ctcagggcac ccccaggtgg tgcggaaagc caggcagttg
ggacagaggt gcccacgagg 1532gcagaggccg gtgctaaggg gatggggaag
aagggacaag attcccagag aggagaggag 1592gctgttggta ggaaagtggc
agggctgggg gagacccagc cccaagggtc cggggcggag 1652gatgctttgt
tcttttctgg ttttggttcc tctttcgcgg ggggtggggg aggtcaacag
1712ggactgagtg gggcagaggc ccagaagtgc cagcctgggg agccgtttgg
gggcagcccc 1772ttctgcccac cccatccttc ttcctctcca gagatgccag
gggggcgtgt atgctctgcc 1832ccttccctca gacaggggct gggtggggag
gctctttagg ctcaggagaa gcattttaaa 1892gaaaccccca ccctgccgcc
cgcattataa acacaggaga ataatcaata gaataaaagt 1952gaccgactgt
caaaaaaaaa aaa 19752353PRTHomo sapiens 2Met Ser Lys Pro Pro Asp Leu
Leu Leu Arg Leu Leu Arg Gly Ala Pro1 5 10 15Arg Gln Arg Val Cys Thr
Leu Phe Ile Ile Gly Phe Lys Phe Thr Phe 20 25 30Phe Val Ser Ile Met
Ile Tyr Trp His Val Val Gly Glu Pro Lys Glu 35 40 45Lys Gly Gln Leu
Tyr Asn Leu Pro Ala Glu Ile Pro Cys Pro Thr Leu 50 55 60Thr Pro Pro
Thr Pro Pro Ser His Gly Pro Thr Pro Gly Asn Ile Phe65 70 75 80Phe
Leu Glu Thr Ser Asp Arg Thr Asn Pro Asn Phe Leu Phe Met Cys 85 90
95Ser Val Glu Ser Ala Ala Arg Thr His Pro Glu Ser His Val Leu Val
100 105 110Leu Met Lys Gly Leu Pro Gly Gly Asn Ala Ser Leu Pro Arg
His Leu 115 120 125Gly Ile Ser Leu Leu Ser Cys Phe Pro Asn Val Gln
Met Leu Pro Leu 130 135 140Asp Leu Arg Glu Leu Phe Arg Asp Thr Pro
Leu Ala Asp Trp Tyr Ala145 150 155 160Ala Val Gln Gly Arg Trp Glu
Pro Tyr Leu Leu Pro Val Leu Ser Asp 165 170 175Ala Ser Arg Ile Ala
Leu Met Trp Lys Phe Gly Gly Ile Tyr Leu Asp 180 185 190Thr Asp Phe
Ile Val Leu Lys Asn Leu Arg Asn Leu Thr Asn Val Leu 195 200 205Gly
Thr Gln Ser Arg Tyr Val Leu Asn Gly Ala Phe Leu Ala Phe Glu 210 215
220Arg Arg His Glu Phe Met Ala Leu Cys Met Arg Asp Phe Val Asp
His225 230 235 240Tyr Asn Gly Trp Ile Trp Gly His Gln Gly Pro Gln
Leu Leu Thr Arg 245 250 255Val Phe Lys Lys Trp Cys Ser Ile Arg Ser
Leu Ala Glu Ser Arg Ala 260 265 270Cys Arg Gly Val Thr Thr Leu Pro
Pro Glu Ala Phe Tyr Pro Ile Pro 275 280 285Trp Gln Asp Trp Lys Lys
Tyr Phe Glu Asp Ile Asn Pro Glu Glu Leu 290 295 300Pro Arg Leu Leu
Ser Ala Thr Tyr Ala Val His Val Trp Asn Lys Lys305 310 315 320Ser
Gln Gly Thr Arg Phe Glu Ala Thr Ser Arg Ala Leu Leu Ala Gln 325 330
335Leu His Ala Arg Tyr Cys Pro Thr Thr His Glu Ala Met Lys Met Tyr
340 345 350Leu
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